JP2011178680A - Antiallergic agent derived from adzuki bean and food containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel antiallergic agent derived from an adzuki bean exhibiting higher medicinal effects than existing antiallergic medicines in perception of unutilized effective components of an adzuki bean, and a food containing the same. <P>SOLUTION: The antiallergic agent derived from an adzuki bean is obtained by separating a supernatant liquid from broth obtained by boiling an adzuki bean in boiling water, causing the supernatant liquid to adsorb on a synthetic adsorbent, and subsequently adding 10-40% ethanol aqueous solution to the synthetic adsorbent after the adsorption to elute the extracts, and exhibits activities on a type I allergic reaction. The food is obtained by adding the antiallergic agent derived from an adzuki bean thereto. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  TECHNICAL FIELD The present invention relates to an azuki bean-derived antiallergic agent and a food containing the same, and more particularly to an antiallergic agent derived from a hot water extract of azuki bean and a food to which the antiallergic agent is added.

  Typical symptoms of allergic diseases are allergic rhinitis, bronchial asthma, atopic dermatitis, food allergy, etc. caused by pollen such as cedar and cypress. At present, in these treatments and symptom improvement, mediator release inhibitors such as antihistamines, antithromboxane drugs (TXA2 antagonists, thromboxane A2 receptor antagonists), leukotriene antagonists (RTs antagonists), Th2 cytokines Inhibitors are prescribed. Although the above-mentioned pharmaceuticals have a certain effect on symptom improvement, there are side effects such as causing general malaise.

  Thus, in recent years, many attempts have been reported to use natural product-derived components to improve symptoms of allergic diseases. For example, there are anti-allergic agents (Patent Documents 1 and 2 etc.) containing an extract of strawberry tea (Rubus suavissims S. Lee). In addition, extracts of cedar (Cryptomeria japonica D. Don) leaves (Patent Document 3), anti-allergic agents of catechins extracted from green tea leaves such as “Benifuuki” and “Benifuji” (Patent Document 4 etc.) There is. It is an attempt to make effective use of these natural products in daily life to help suppress or reduce allergic reactions while relatively suppressing side effects.

  Apart from the above-mentioned circumstances, the inventor has been conducting various studies focusing on azuki. Azuki beans (Vigna angularis) are processed softly by heating mainly by boiling (cooking), and are processed into food ingredients for cooking such as red rice or confectionery ingredients such as “Ogura An” with added sugar. When processing azuki bean, a large amount of azuki soup is produced. It is known that azuki bean broth contains polyphenols, saponins, anthocyanins, and glycosides bound to sugar molecules. However, it is often discarded as it is without being used.

  The following studies have been reported as an effective use of the bioactive ingredients contained in the azuki bean broth. For example, antitumor activity has been reported for glycosides obtained from azuki bean broth (see Patent Document 5, Non-Patent Documents 1 and 2, etc.). Moreover, about the physiological effect of the alcohol extract of azuki bean broth, effects, such as a hypoglycemic activity, cell adhesion inhibition, hyperlipidemia prevention, etc. are reported (refer nonpatent literature 3, 4, 5, 6 etc.). .

  And the inventor who repeated the earnest research about the bioactivity effect of an antiallergy regarding the component contained in azuki bean came to obtain the knowledge that the component contained in azuki bean is useful also for allergic diseases.

Japanese Patent No. 2700958 JP 2003-169631 A JP 2007-254322 A Japanese Patent No. 4272691 JP 2003-63967 A

Ito Tomohiro et al., Japanese Journal of Food Science and Technology Vol. 49, No. 5, p. 339-344 (May 2002) Tomohiro Ito et al., Journal of Japanese Society of Nutrition and Food, Vol.58, No.5, p. 281-287 (2005) T.A. Itoh et al. , Biosci. Biotechnol. Biochem. 68 (12), 2421-2426 (2004). T.A. Itoh et al. , Biosci. Biotechnol. Biochem. 69 (3), 448-454 (2005) T.A. Itoh et al. , Nutrition, 25, 134-141 (2009) T.A. Itoh et al. , Nutrition, 25, 318-321 (2009)

  The present invention has been made in view of the above points, and focuses on an unused active ingredient of azuki bean, and contains an azuki bean-derived antiallergic agent having a higher efficacy than new and existing antiallergic drugs, and this To provide food.

  That is, the invention of claim 1 separates a supernatant from boiled azuki bean boiled with hot water, adsorbs the supernatant to a synthetic adsorbent, and then adds an aqueous ethanol solution to the adsorbed synthetic adsorbent In addition, the present invention relates to an azuki bean-derived antiallergic agent characterized by eluting the extract.

  Invention of Claim 2 concerns on the azuki bean origin antiallergic agent of Claim 1 whose said ethanol aqueous solution is 10%-40% ethanol aqueous solution.

  The invention of claim 3 relates to the azuki bean-derived antiallergic agent according to claim 1 or 2, wherein the extract exhibits activity against type I allergic reaction.

  The invention of claim 4 relates to a food containing azuki bean-derived antiallergic agent, characterized in that the azuki bean-derived antiallergic agent according to any one of claims 1 to 3 is added to the food.

  According to the azuki bean-derived antiallergic agent of the invention of claim 1, after separating the supernatant from the broth produced by boiling the azuki bean in hot water, adsorbing the supernatant to the synthetic adsorbent, Since an aqueous ethanol solution was added to the synthetic adsorbent and the extract was eluted, a new azuki bean-derived antiallergic agent could be obtained by focusing on the unused active ingredients of azuki bean that had been discarded so far.

  According to the azuki bean-derived antiallergic agent according to the invention of claim 2, in the invention of claim 1, since the ethanol aqueous solution is 10% to 40% ethanol aqueous solution, it has higher efficacy than existing antiallergic agents. Azuki bean-derived antiallergic agent could be obtained.

  According to the azuki bean-derived antiallergic agent according to the invention of claim 3, in the invention of claim 1 or 2, the extract exhibits activity against type I allergic reaction, which can contribute to improvement of symptoms of type I allergy.

  According to the azuki bean-derived antiallergic agent-containing food according to the invention of claim 4, the azuki bean-derived antiallergic agent according to any one of claims 1 to 3 is added to the food. Ingestion becomes possible.

It is a schematic process drawing which obtains the azuki bean origin antiallergic agent of the present invention. It is a schematic diagram of an intracellular signal transduction model. It is a graph of the degranulation reaction inhibitory rate of four types of extracts derived from azuki bean broth. It is a graph of the degranulation reaction inhibitory rate of azuki bean broth extract, strawberry tea extract, and cromoglycate sodium. It is a graph of the intracellular calcium ion concentration regarding azuki bean origin extract. It is a graph of the intracellular calcium ion concentration regarding cromoglycate sodium. It is a graph of intracellular ROS production after adding azuki bean-derived extract. It is a graph of intracellular ROS production after DPI addition. It is a comparison photograph of the Western blot of the protein of a cell membrane fraction and a cytoplasm fraction. It is the graph which showed antioxidant activity. It is the 1st detection electrophoresis photograph of each phosphorylated protein of the presence or absence of antigen stimulation, and an adzuki origin extract addition. It is the 2nd detection electrophoretic photograph of each phosphorylated protein of the presence or absence of antigen stimulation, and azuki bean origin extract addition. It is the photograph of the peeling skin of the mouse | mouth which administered the negative control, the positive control, and the boiled powder of azuki bean, and performed PCA reaction. It is the photograph of the peeling skin of the mouse | mouth which administered the extract derived from azuki bean and the tea extract, and performed PCA reaction. It is the graph which digitized the blood vessel permeation enhancement inhibitory effect in the mouse | mouth of FIG.13 and FIG.14.

  About the azuki bean origin antiallergic agent of this invention, the process of obtaining this is demonstrated using the schematic process drawing of FIG. First, azuki bean (Vigna angularis) is boiled (cooked) for 20 to 60 minutes in hot water at 90 to 95 ° C. in a steaming kettle or the like. After the heating, azuki bean and hot water (boiled juice) are separated. Azuki bean is added to sugar and processed into Ogura An.

  The broth produced when the adzuki beans are boiled is cooled to room temperature, and the broth is divided into a supernatant and a precipitate. In addition to filter filtration, centrifugal separation under the conditions of Examples described later is also used for separating the broth.

  Various hot water soluble components are contained in the supernatant of the broth. Therefore, the supernatant is once adsorbed to the synthetic adsorbent for concentration and elution separation of the components in the supernatant. The synthetic adsorbent is an appropriate resin column, resin beads, or the like. In the examples described later, beads having a particle diameter of 0.25 mm or more provided with pores made of styrene-divinylbenzene resin were used.

  An aqueous solution of ethanol is added to the synthetic adsorbent adsorbing various components in the supernatant and sent. The adzuki bean extract adsorbed on the synthetic adsorbent is eluted. Thus, the component obtained by the process of azuki bean boiling, supernatant separation, adsorption, and elution is the azuki bean-derived antiallergic agent defined in the present invention. The presence or absence of an elution component present in the ethanol aqueous solution can be easily grasped by confirming the absorbance at a predetermined wavelength (280 nm).

  The aqueous ethanol solution is prepared as a solution (v / v%) with a concentration gradient (gradient) in which the ethanol concentration is gradually increased gradually from the state of water, and is used for elution of the extract. In the examples, the concentration of the aqueous ethanol solution (v / v%) is 4% from the initial 0% to the 10% to 40% concentration solution, the 40% to 60% concentration solution, and the 60% to 80% concentration solution. It's kind. In terms of the accuracy of forming the concentration gradient, it is roughly divided into the concentration regions. Note that when the synthetic adsorbent is dried, the separation performance fluctuates, so that a necessary amount of water is passed when the concentration is switched.

  By evaporating and drying moisture from the elution fraction (fraction) of each ethanol aqueous solution concentration, an extract sorted according to the concentration of the ethanol aqueous solution can be obtained. This extract is azuki bean-derived antiallergic agent. In the concentration range of the four types of ethanol aqueous solutions, the antiallergic properties are uniformly expressed. Among them, the expression of anti-allergic properties of ethanol aqueous solution having a concentration range of 10% to 40% is remarkable. Since the extract is a component derived from azuki bean broth, it is assumed that polyphenol components such as glycosides, pigment components, and various catechins are included. Therefore, the azuki bean-derived antiallergic agent of the present invention is considered to be a mixture containing a plurality of components such as the aforementioned glycoside.

  The azuki bean-derived antiallergic agent of the present invention is a medicinal ingredient that is considered useful for the treatment and alleviation of allergic symptoms. In addition, it is a medicinal ingredient that is said to be effective in improving the quality of daily life (QOL: Quality of Life). When used as a drug, the dosage form for oral administration is appropriate as tablets, capsules, powders, granules, liquids and the like. Moreover, it can also be set as an injection, an instillation, a spray, a nasal drop, an eye drop etc. In the listed drugs, azuki bean-derived antiallergic agents may be used alone or in combination with other drugs.

  Here, allergies and symptoms will be briefly described according to their types. Generally, allergies are classified into the following types I to IV.

[Type I allergy (immediate type)]
IgE (immunoglobulin E) secreted from B cells by signals such as IL-4 binds to the receptor FcεRI on the surface of mast cells and basophils. When various allergens (antigens) bind to IgE, chemical mediators such as histamine, serotonin, and leukotriene are secreted from mast cells and basophil cells. The various chemical mediators cause symptoms such as swelling and rhinitis due to vasodilation, asthma due to bronchoconstriction, urticaria and atopic dermatitis. Symptoms of food allergy and anaphylactic shock are also included in type I allergy.

[Type II allergy (cytotoxic type)]
When self cells are recognized as antigens, antibodies such as IgG and IgM against these cells are produced. This is a reaction in which an antigen-antibody reaction attacks between self cells. Symptoms include autoimmune solution anemia, Hashimoto's disease, and Graves' disease.

[Type III allergy (Arsus type)]
An immune complex is formed by the reaction between the soluble antigen and IgG. This is a reaction resulting from tissue injury caused by this immune complex. Symptoms include rheumatoid arthritis and systemic lupus erythematosus.

[Type IV allergy (delayed type)]
Activation of Th1 cells and Th2 cells causes production of cytokines such as IL-1, IFN-γ, and IL-5, and there is a reaction when foreign substances are processed by macrophages, neutrophils, and NK cells. Symptoms include atopic dermatitis and tuberculin reaction. In particular, in the case of atopic dermatitis after the progression of type I allergy, the inflammation tends to worsen.

  The azuki bean-derived antiallergic agent of the present invention is considered to be effective in suppressing the allergic reaction. In particular, it is presumed that the effect of suppressing the type I allergic reaction is high, as can be understood from the intracellular mechanism of action disclosed in the examples described later. Type I allergies develop serious symptoms that require urgency, and many symptoms that cause discomfort in daily life such as asthma, runny nose, and sneeze. Therefore, if you can sensitize mast cells and basophil cells all the time by ingesting antiallergic agents on a daily basis and alleviate the symptoms, you can improve the quality of life and disease caused by lifestyle habits. Greatly contribute to

  The antiallergic agent of the present invention is a natural product and is an extract derived from azuki bean that has been edible for a long time. Therefore, the azuki bean-derived antiallergic agent of the present invention is familiar with food. Therefore, azuki bean-derived antiallergic agent-containing food newly added with azuki bean-derived antiallergic agent can be prepared.

  Foods to which Azuki-derived antiallergic agents (Azuki bean juice extract) can be added are specifically Mizuyokan, Yokan, Ogura An, Dorayaki, Azuki Mousse, Ice Cream, Candy, Caramel, Custard Pudding, Coffee jelly, gummy, bavaroa, marshmallow, castella, hot cake, cookies, fruit juice, carbonated drinks, etc. Furthermore, the tablet used as a supplement is also mentioned.

  When manufacturing the food containing azuki bean-derived antiallergic agent listed, the content of the azuki bean-derived antiallergic agent is the taste of the food itself, the change in taste after addition, other additive ingredients, the amount of food consumed per time, the food consumption It is stipulated by comprehensively considering the frequency, seasonality, sales form, and the consumer group taking account of age, gender, and occupation. The amount of the azuki bean-derived antiallergic agent added to the food is arbitrary, and may be, for example, 0.01% to 100% by weight (that is, the whole amount of the azuki bean-derived antiallergic agent), 1% to 80% by weight. .

  The inventors extracted azuki bean-derived antiallergic agent from azuki bean according to the following methods and procedures. And the confirmation of the action mechanism in the cell of an antiallergic agent and the expression in the living body were confirmed.

[Preparation of extract derived from azuki bean broth]
Azuki beans from Tokachi, Hokkaido were used as the raw material for azuki beans. Azuki beans were put into an open-type steaming kettle, heated from water and boiled for 45 minutes. The boiled and softened azuki bean and its broth were separated. The broth was cooled to near room temperature (about 20 ° C.), and then centrifuged at 16500 × g (9000 rpm) for 10 minutes. To 1 kg of the supernatant obtained by centrifugation, 300 g of an aromatic synthetic adsorbent (reverse phase adsorbent “DIAION HP-20” manufactured by Mitsubishi Chemical Corporation) was added and stirred at 4 ° C. for 12 hours. After dividing into a synthetic adsorbent and other liquids, the synthetic adsorbent was washed with distilled water and further eluted several times with 40% ethanol aqueous solution (v / v).

  The washed and eluted synthetic adsorbent was packed in a column (inner diameter 5 mm × total length 300 mm). A plurality of the synthetic adsorbent packed columns were prepared. First, only distilled water was sent to the column packed with the synthetic adsorbent, and a solution was prepared while gradually increasing the ethanol ratio. In the present embodiment, the elution of only distilled water from the beginning is carried out according to four types of concentrations: an aqueous ethanol solution of 10% to 40% concentration, an aqueous ethanol solution of 40% to 60% concentration, and an aqueous ethanol solution of 60% to 80% concentration. Elution was performed. Ethanol subjected to elution is accompanied by a concentration gradient (gradient). For this reason, when the density is switched, a range of the density inevitably occurs. Thus, elution conditions having a concentration range were used as described above.

  For each of elution from distilled water only, 10% to 40% ethanol aqueous solution, 40% to 60% ethanol aqueous solution, and 60% to 80% ethanol aqueous fraction (fraction). (Shibata Kagaku Co., Ltd.) was used to evaporate and dry the water in the fraction. Thus, four kinds of extracts derived from azuki bean broth with different elution conditions were obtained. In the following description and drawings, the extract obtained from the elution of distilled water alone is referred to as “WEx”, and the extract obtained from the elution of an aqueous ethanol solution having a concentration of 10% to 40% is referred to as “EtEx.40”, 40% to 60%. The extract obtained from the elution of the aqueous ethanol solution with a concentration of% is denoted as “EtEx.60”, and the extract obtained from the elution of an aqueous ethanol solution with a concentration of 60% to 80% is denoted as “EtEx.80”.

[Verification of intracellular mechanism]
The inventor who prepared the extract derived from four different kinds of azuki bean broth revealed the presence or absence of antiallergic performance in the cell and the action site in the cell, thereby improving the medicinal efficacy of the extract derived from the bean broth. confirmed. Hereinafter, verification experiments will be described in order.

<1. Inhibition of degranulation reaction>
The degranulation reaction inhibitory action was measured as an index of antiallergic performance for extracts derived from four types of azuki bean broth with different elution conditions. At the same time, we tried to compare with existing drugs that exert anti-allergic effects. The degranulation reaction inhibitory action is caused by β-hexosaminidase (chemical mediator), which is a chemical mediator, along with the degranulation in the schematic diagram of the intracellular signal transduction model such as basophil cells disclosed in FIG. The secretion inhibitory effect of β-hexosaminidase) was used as an index.

Rat basophil cells RBL-2H3 were suspended in MEM medium supplemented with 10% FBS at a concentration of 5.0 × 10 ⁵ cells / mL. Mouse monoclonal anti-dinitrophenyl group IgE antibody (anti-DNP-IgE antibody) was added to the same medium at a concentration of 200 ng / mL, and the rat cells were sensitized with the antibody for 24 hours. After completion of the culture, the medium was removed by aspiration, washed twice with a Siraganian buffer, and replaced with 160 μL of the same buffer.

  In the preparation of the chemicals for addition, first, four types of extracts derived from azuki bean broth, WEx, EtEx. 40, EtEx. 60, and EtEx. 80 and, as a comparison, the tea extract (hereinafter referred to as Rubus ext.) Was dissolved in dimethyl sulfoxide (hereinafter referred to as DMSO). The extract was diluted with a silaganian buffer until the final concentration of DMSO in which each extract was dissolved was 0.1% or less. As a positive control, DMSO alone was diluted with Siraganian buffer. In addition, as a drug used as an antiallergic agent, the drug name “interle” and the molecular name “sodium cromoglycate” (hereinafter referred to as Dscg) are used, and this Dscg is also dissolved in DMSO and extracted. Dilute with Silaganian buffer as in

  Extracts derived from azuki bean broth at various concentrations after dilution with buffer solution, strawberry tea extract, positive control, and 20 μL of Dscg drug solution were added to the rat cell culture medium after antibody sensitization and incubated at 37 ° C. for 30 minutes. . Subsequently, 20 μL of dinitrophenylated bovine serum albumin (DNP-BSA) was added, and antigen stimulation was performed at 37 ° C. for 30 minutes. Thereafter, the reaction was stopped by allowing to stand for 10 minutes under ice cooling. 10 μL of the supernatant was collected from each medium and transferred to a 96-well plate. 50 μL of p-nitrophenyl-N-acetyl-β-D-glucosamide (p-NAG) solution was added to each well of the plate and allowed to develop color at 37 ° C. for 1 hour. After completion of the reaction, 200 μL of the reaction stop solution was added, and the absorbance “A” at 405 nm was measured with a microplate reader.

The above positive control was treated in the same manner, and the absorbance “B” was determined. In addition, the absorbance “C” of the negative control without antigen stimulation, in which only the Siraganian buffer was added instead of the DNP-BSA, was also determined. Based on each absorbance, the β-hexosaminidase release inhibition rate (P _i ) (unit:%) was determined by the following formula (i).

Furthermore, the direct inhibitory activity of β-hexosaminidase by the sample was calculated. Accordingly, from the β-hexosaminidase enzyme solution prepared from rat basophil cells RBL-2H3 and the sample solution used for measuring the degranulation reaction, the enzyme direct inhibitory activity rate (Q _i ) (unit:%) was determined. The absorbance of the sample treatment is “D”, and the absorbance of the positive control is “E”. From the enzyme direct inhibition activity rate (E _i ) and release inhibition rate (F _i ) determined from both formulas (i) and (ii), the degranulation reaction inhibition rate (R _i ) (unit:%) according to formula (iii) Asked.

  The graph of FIG. 3 shows four types of extracts derived from azuki bean broth, WEx, EtEx. 40, EtEx. 60, and EtEx. 8 is a result of degranulation reaction inhibition rate (%) concerning 80. The units 50 μg / mL and 100 μg / mL in the graph are concentrations in the medium used for the experiment. From these results, suppression of the degranulation reaction was revealed under any elution conditions. The reaction activity is concentration-dependent. Of these, EtEx. It was revealed that an extract derived from azuki bean juice obtained from elution of an aqueous ethanol solution having a concentration of 40, that is, 10% to 40% has the highest activity.

  The graph of FIG. 4 shows EtEx. 40 is a comparison result of the degranulation reaction inhibition rate (%) between strawberry tea extract (Rubus ext.) And cromoglycate sodium (Dscg). The units 50 μg / mL and 100 μg / mL in the graph are concentrations in the medium used for the experiment. This result shows that the inhibitory effect of azuki bean-derived extract (EtEx.40) is superior to existing natural product extracts and pharmaceuticals.

<2. Changes in intracellular calcium ion concentration>
It is believed that the degranulation reaction in type I allergy involves an increase in intracellular calcium ion concentration due to the inflow of calcium ions (Ca ²⁺ ). As shown in the schematic diagram of FIG. 2, a calcium ion channel is opened by reactive oxygen species (H ₂ O ₂ etc.), and the calcium ion flows from outside the cell into the cell, and the signal transduction via IP3 is small. The pathway by which calcium ions are released from the endoplasmic reticulum ER is now known. Therefore, the inventor examined “EtEx.40”, which is an extract derived from azuki bean broth having the highest inhibitory effect on the degranulation reaction, and verified the influence on the inflow of calcium ions.

  The intracellular calcium concentration was measured using “Calcium Kit-Fluo 3” manufactured by Dojindo Laboratories. A 96-well microplate was seeded with RBL-2H3 cells treated with IgE infection, 100 μL of a loading buffer containing 3 μM of the fluorescent indicator Fluo3-AM was added to each well, and the fluorescent indicator was incorporated at 37 ° C. for 30 minutes. . After 30 minutes, excess fluorescent indicator was washed away by washing twice with a phosphate buffer (PBS (−)). After washing, 80 μL of Recording buffer and EtEx. Of Azuki-derived extract diluted with DMSO and Siraganian buffer were prepared. 40 μl or 10 μL each of the control drug Dscg diluted with DMSO and Siraganian buffer was added and treated for 30 minutes.

  The treated microplate was attached to a microplate reader for fluorescence measurement, and 10 μL of DNP-BSA was added as an antigen to initiate the reaction. While maintaining 37 ° C., the fluorescence intensity by the fluorescent indicator for 4 minutes was measured every 5 seconds. EtEx. Of azuki bean-derived extract. FIG. 5 shows the transition result of the intracellular calcium ion concentration with respect to 40, and FIG. 6 shows the transition result of the control drug Dscg. Ethylene glycol tetraacetate (EGTA) in both graphs is a chelating agent that captures calcium ions and was used for comparison.

  Regarding FIG. 5, the calcium ion concentration in the broken line plotted with a black circle is a reaction accompanied by antigen stimulation (DNP-BSA administration) (Ag (+) in the graph). The white circle and the broken line of the plot are reactions without antigenic stimulation (Ag (−) in the graph). The broken line of the plot in the black triangle is the reaction in which antigen stimulation was performed by adding EGTA. Obviously, calcium ions are chelated, so the amount is lower than the black circle. Next, the black square and the polygonal line of the plot are EtEx. This is a reaction in which 40 was added to perform antigen stimulation. EtEx. The addition of 40 exerts an inhibitory effect on intracellular calcium ion concentration. Furthermore, EtEx. No. 40 maintains the intracellular calcium ion concentration on the lower side as compared with the state in which there is no antigen stimulation or EGTA addition.

  In FIG. 6 as a control, the broken line of the black circle, white circle, and black triangle plots is the result of the same processing as in FIG. The polygonal line in the black square is the reaction in which Dsgg was added to stimulate the antigen. Although Dscg suppresses an increase in calcium ion concentration as compared with a state in which antigen stimulation is present, it can be said that the activity of inhibiting the increase in calcium ion concentration is weak. Based on the results of both figures, EtEx. No. 40 expresses a very strong activity for suppressing the increase of intracellular calcium ions.

<3. Inhibition of intracellular ROS production>
In the living body, “ROS”, which is a reactive oxygen species such as superoxide anion radical, hydroxy radical, hydrogen peroxide, singlet oxygen, is constantly produced. The amount of ROS produced by leukocytes, particularly neutrophils, is significant. ROS is known to be a signaling substance for inducing immune responses such as bactericidal and foreign substance removal. In addition, ROS is thought to induce the increase in intracellular calcium ion concentration described above, and to cause a degranulation reaction in this connection. For example, the pathway of calcium ion channel opening and calcium ion inflow by ROS such as H ₂ O ₂ shown in the schematic diagram of FIG. The measurement of the ROS concentration was examined on the basis of the inhibitory activity on the increase of intracellular calcium ion found from the results in “2. Variation in intracellular calcium ion concentration”. Thus, an attempt was made to measure the intracellular ROS concentration after antigen stimulation using a DCF fluorescent indicator that specifically detects reactive oxygen species.

A membrane-permeable probe 2 ′, 7′-Dichlorodihydrofluorescein-diacetate (hereinafter referred to as DCFH-DA) migrates from the outside of the cell into the cell and is hydrolyzed by esterase in the cytoplasm to become 2 ′, 7′- It changes to Dichlorodifluorescein (hereinafter referred to as DCFH). DCFH remains intracellular because it loses membrane permeability. DCFH reacts with intracellular reactive oxygen species (ROS) such as hydrogen peroxide (see H ₂ O _{2 in} FIG. 2) and is oxidized to 2 ′, 7′-Dichlorofluorescein (hereinafter referred to as DCF). . Since DCF emits fluorescence, the intracellular oxidation state (the amount of ROS) can be quantified by measuring the DCF fluorescence intensity of cells administered with DCFH-DA. When an antioxidant substance is present in the cell, the oxidation of DCF is suppressed, and the antioxidant activity of the test substance in the cell can be evaluated by reducing the fluorescence intensity.

  RBL-2H3 cells were cultured, treated with 10 μM DCFH-DA fluorescent dye for 30 minutes, and incorporated into the cells. After 30 minutes, the fluorescent dye that was not taken up into the cells was washed with PBS (−), and then the extract of azuki bean-derived extract, EtEx. 40, each of the NADPH oxidase inhibitor diphenyleneneodium chloride (hereinafter referred to as DPI), the positive control, and the negative control were treated in the same manner as the above-mentioned “1. Deagglomeration reaction inhibitory action”. After the antigen treatment, the fluorescence intensity was measured over time for 10 minutes at 1 minute intervals.

  The graph of FIG. 7 shows EtEx. 40 shows the transition after the addition of 40, and the black line in the plot shows the EtEx. It is a time-dependent change at the time of performing antigen stimulation by adding 40. FIG. The graph of FIG. 8 shows the transition after administration of DPI, and the broken line of the plot in black squares is the change over time when antigen stimulation was performed by adding DPI. In both graphs, a black circle and a broken line of the plot are reactions accompanied by antigen stimulation (DNP-BSA administration) (Ag (+) in the graph). The white circle and the broken line of the plot are reactions without antigenic stimulation (Ag (−) in the graph).

  As shown in the figure, the effect of DPI on suppressing intracellular ROS production is clear as compared with the positive control. On top of that, EtEx. 40 was found to significantly suppress ROS production more than DPI. It was also revealed that ROS production was suppressed more than the negative control.

<4. NADPH oxidase inhibitory activity>
Calcium channels that regulate the influx of calcium ions into the cell are present in the cell membrane. This calcium channel is regulated by intracellular ROS (see above). It has been reported that NADPH oxidase (hereinafter referred to as NOX) is involved in intracellular ROS production after antigen stimulation in mast cells. The activation of NOX will be briefly described with reference to the schematic diagram of FIG. First, Rac subunit p40 ^phox present in the cytoplasm, p47 ^phox, p67 linked to form a tetramer with ^phox. Next, ROS is produced when this tetramer moves to the cell membrane subunits gp91 ^phox and p22 ^phox . Therefore, by examining the membrane fraction and the cytoplasm fraction, the NOX activity can be grasped as a result of subunit transfer.

  Upon separation of the membrane fraction and the cytoplasm fraction, a Proteo Extract Subcellular Proteome Extraction kit (code 353790) manufactured by Calbiochem was prepared. FIG. 9 is a photograph of a Western blot of proteins of the cell membrane fraction and the cytoplasm fraction. From left, “Ag (−)” without negative stimulation as a negative control, “Ag (+)” with DNP-BSA administration as a positive control, EtEx. The order is 40 additions.

In the presence of antigen stimulation (Ag (+)), p40 ^phox , p67 ^phox , p47 ^phox , and Rac could be detected from the cell membrane side, so that the migration to the membrane side is clear. On the other hand, EtEx. When 40 is added, ^p67phox and Rac are hardly detected from the cell membrane side. Therefore, it was possible to confirm that the subunits present in the cytoplasm (p40 ^phox , p47 ^phox , p67 ^phox , and Rac) were collectively suppressed to the membrane side.

<5. Antioxidant activity>
It is also speculated that the intracellular ROS concentration decreases due to the antioxidant activity of the added sample itself. Therefore, it is necessary to clarify the antioxidant activity of the sample itself. The radical scavenging ability of each sample was measured in vitro by the radical scavenging ability of 2,2-Diphenyl-1-picrylhydrazyl (hereinafter referred to as DPPH), which is widely used as a radical generator. DPPH has a maximum absorption of 517 nm in the radical state. By being reduced by an antioxidant, the absorbance at 517 nm decreases. By measuring the decrease in absorbance, the antioxidant activity of the sample (compound) can be measured.

  DPPH was dissolved in a 50% ethanol aqueous solution to prepare a 250 μM solution, filtered through a 0.22 μm filter to remove insoluble matters, and used. As a sample, an extract of azuki bean derived EtEx. 40, Vitamin C (L-ascorbic acid), Vitamin E (α-tocopherol), and Trolox were used for conversion. These were dissolved in DMSO and diluted with 50% ethanol. 20 μL of the sample solution was dispensed into a 96-well microplate. To this, 180 μL of DPPH solution was added and reacted at room temperature for 20 minutes. After the reaction, the absorbance at 517 nm was measured.

  The graph of FIG. 10 shows EtEx. It is the result of DPPH radical scavenging ability of 40 (black circle plot), vitamin C (black triangle plot), and vitamin E (black square plot). EtEx. Of azuki bean-derived extract. No. 40 shows a high antioxidant activity three times or more that of vitamins C and E. From the knowledge so far, EtEx. It is speculated that the suppression of intracellular ROS production on which 40 acts is due to both NADPH oxidase inhibitory activity and antioxidant activity.

<6. Quantitative comparison of phosphorylated proteins>
As can be understood from the schematic diagram of FIG. 2, it is becoming clear that phosphorylation of various proteins occurs upstream of the degranulation reaction, causing factors such as cytokine gene expression and arachidonic acid cascade. Therefore, EtEx. It was investigated whether 40 actually acts on suppression of phosphorylation.

  Similar to the aforementioned “1. degranulation reaction inhibitory action”, EtEx. Forty treatments and antigen-stimulated cells were prepared. In preparing the cell extract, the cells were washed twice with ice-cold PBS (−). Prepare a buffer containing 10 mM Tris-HCl buffer (pH 7.5), 1 mM EDTA, 1% NP-40, 0.1% Sodium Deoxychlorate, 150 mM NaCl, 0.1% SDS. Then, ultrasonic homogenization was performed on ice with the same buffer. The lysate was centrifuged at 20000 × g (14500 rpm) at 4 ° C. for 3 minutes. The obtained supernatant was used as a soluble fraction. About each cell extract, protein concentration was measured by BCA method (bicinchoninic acid method), and it diluted so that it might become equal concentration. Cell lysates were separated by SDS-PAGE and transferred to PVDF membrane. The PVDF membrane was blocked with 5% skim milk and probed with various antibodies. Next, a horseradish peroxidase-conjugated secondary antibody was reacted, and color was developed using an ECL method (Enhanced Chemiluminescence method).

11 and FIG. 12 show the negative control “Ag (−)” without antigen stimulation, the positive control with antigen stimulation (administration of DNP-BSA) “Ag (+)”, EtEx of azuki bean-derived extract. . It is a detection result of each phosphorylated protein shown in the order of 40 addition. Results of “Lyn” and “p-Lyn”, “Syk” and “p-Syk”, “cPLA ₂ ” and “p-cPLA ₂ ”, “Akt” and “p-Akt”, and “PLCγ1, PLCγ2” From the extract of azuki bean-derived extract, EtEx. About the cell which added 40, it turned out that the phosphorylation of Lyn, Syk, PLC (gamma) 1, PLC (gamma) 2 was strongly suppressed mainly. Therefore, EtEx. 40 can be said to act upstream of the signal transduction system in the mechanism of action of allergic reactions (see FIG. 2).

<7. Inhibition of hypervascular permeability>
In the in vitro system described so far, the inventor has convinced that the extract derived from azuki bean (EtEx. 40) of the present invention is effective in suppressing allergic reactions. Therefore, the inventor decided to verify the effect in the in vivo system described below. The PCA reaction (Passive Cutaneous Anaphylaxis Reaction) is a test method for measuring increased vascular permeability, which is one of the symptoms of type I allergy. Evans blue saline, IgE-DNP, and DNP-BSA antigens were administered from the tail vein of mice, and allergic reaction was induced by DNP antigen stimulation. After 1 hour, the animal was killed by anesthesia and the skin was peeled off.

  13 and 14 show a negative control [Negative Control], a positive control [Positive Control], azuki bean juice powder [HWEA (500 mg / kg, 1000 mg / kg per mouse body weight)], azuki bean-derived extract [EtEx. 40 (100 mg / kg, 250 mg / kg, 500 mg / kg per mouse body weight)], and strawberry tea extract [Rubus ext. (1000 mg / kg per mouse body weight)]. Azuki bean broth powder is a dried product of the broth when boiled with hot water. Azuki bean juice powder, azuki bean-derived extract, and strawberry tea extract are the results of skin photographs of mice peeled off after being administered from the tail vein of mice prior to antigen stimulation. For each dose, three mice were prepared for the experiment.

  Since it is expressed in black and white in the drawing, it is difficult to read the difference for each dose. The negative control is the most white state because there is no onset of type I allergic reaction. In contrast, the positive control is the state of expression of type I allergic reaction. The allergic reaction expands the capillaries and exudes Evans Blue, which makes the entire skin bluish. The darkness increases in the picture. Therefore, anti-allergic properties are exhibited when the degree of exudation of Evans blue after administration of each administration (ie, skin depth) is lower than that of the positive control. As a result, the azuki bean-derived extract EtEx. The anti-allergic performance of 40 is excellent, and the dosage of 250 mg / kg and 500 mg / kg is remarkable among them.

  Based on the results of staining in FIGS. 13 and 14, the pigment content was extracted from the peeled skin, and the absorbance at 720 nm was measured with an absorptiometer. Thus, the inhibitory effect on enhancement of vascular permeability in mice after intravenous injection was quantified and evaluated. The result is the graph of FIG. 15, which coincided with the results of the photographs of FIG. 13 and FIG. Azuki broth powder and strawberry tea extract showed almost the same level of inhibitory effect on vascular permeability enhancement. Low concentration of azuki bean extract EtEx. 40 also showed the same effect as these. However, the extract from azuki bean at a concentration of 500 mg / kg EtEx. In the case of 40, the vascular permeability enhancement inhibitory action was remarkably exhibited as compared with any of the administration examples.

  The results obtained so far as well as the in vivo system were also found to provide the antiallergic activity of the extract derived from the azuki bean broth of the present invention. And the inventor was able to obtain the anti-allergic agent derived from Azuki, which is considered to exhibit higher efficacy than existing products. Moreover, based on the experiment of the blood vessel permeability enhancement inhibitory effect, it was also clarified that the extract derived from the azuki bean broth of the present invention has an excellent activity of suppressing type I allergic reaction.

[Application to food]
The extract derived from the azuki bean broth of the present invention is considered to act effectively as an antiallergic agent. For this reason, the azuki bean-derived extract (antiallergic agent) was blended into individual foods and actually cooked and manufactured. Below, the names of foods and beverages, their recipes (compositions): the left column, and the blending ratio (weight percent notation): the right column are disclosed.

<1. Mizuyokan>
Agar 0.40
Water 45.35
Sugar 27.00
Ogura Ann 27.00
Salt 0.10
Azuki bean extract 0.15
(Total) 100.00

<2. Yokan>
Agar 0.65
Water 24.00
Sugar 24.00
Ogura Ann 48.00
Mizuame 3.20
Azuki bean extract 0.15
(Total) 100.00

<3. Anne Ogura>
Azuki 31.00
35.80 sugar
Salt 0.05
Water 33.00
Azuki bean extract 0.15
(Total) 100.00

<4. Dorayaki>
Soft flour 20.20
Super white sugar 17.00
Whole egg 17.50
Honey 2.50
Baking soda 0.20
Water 12.45
Azuki bean extract 0.15
Ogura Ann 30.00
(Total) 100.00

<5. Azuki Bean Mousse>
Koshian 31.00
Powdered gelatin 1.40
Fresh cream 14.20
Egg white 10.50
Sugar 4.30
Water 38.45
Azuki bean extract 0.15
(Total) 100.00

<6. ice cream>
Yolk 11.00
Granulated sugar 17.80
Milk 53.20
Fresh cream 17.80
Vanilla flavor 0.05
Azuki bean extract 0.15
(Total) 100.00

<7. candy>
Mizuame 45.00
54.75 sugar
Azuki bean extract 0.15
(Total) 100.00

<8. caramel>
Mizuame 34.50
Sugar 19.70
Flour 4.85
Unsweetened condensed milk 34.60
Salt 0.20
Shortening 5.90
Azuki bean extract 0.25
(Total) 100.00

<9. Custard Pudding>
Milk 58.90
Egg 24.00
Sugar 17.00
Azuki bean extract 0.10
(Total) 100.00

<10. coffee jelly>
Coffee (liquid) 82.90
Sugar 8.30
Powdered gelatin 1.40
Coffee liqueur 0.30
Water 7.00
Azuki bean extract 0.10
(Total) 100.00

<11. Gummy>
Granulated sugar 39.00
Water 51.85
Gelatin 6.80
Citric acid 0.50
Fruit flavor 1.60
Azuki bean extract 0.25
(Total) 100.00

<12. Bavaroa>
Milk 47.40
Fresh cream 19.00
Egg yolk 12.60
Sugar 19.00
Gelatin 1.90
Azuki bean extract 0.10
(Total) 100.00

<13. marshmallow>
Mizuame 46.30
Sugar 50.90
Gelatin 2.70
Azuki bean extract 0.10
(Total) 100.00

<14. Castella>
Whole egg 34.30
Egg yolk 3.80
Sugar 34.30
Honey 4.80
Rice bran 1.90
Soft flour 18.00
Water 2.80
Azuki bean extract 0.10
(Total) 100.00

<15. Pancake>
Egg 19.70
Sugar 13.40
Milk 23.40
Baking powder 1.80
Soft flour 36.00
Butter 5.40
Salt 0.15
Vanilla flavor 0.05
Azuki bean extract 0.10
(Total) 100.00

<16. cookie>
Margarine 12.20
Shortening 12.20
Sugar 18.40
Whole egg 8.20
Weak flour 44.80
Powerful powder 4.10
Azuki bean extract 0.10
(Total) 100.00

<17. fruit juice>
Fruit juice 50.00
Fructose dextrose liquid sugar 10.00
Citric acid 0.30
Fruit flavor 0.20
Water 39.25
Azuki bean extract 0.25
(Total) 100.00

<18. Carbonated drink>
Fructose glucose liquid sugar 11.00
Citric acid 0.20
Sodium citrate 0.05
Fruit flavor 0.20
Carbonated water 88.45
Azuki bean extract 0.10
(Total) 100.00

<19. tablet>
Cellulose 30.00
Calcium stearate 2.50
Silicon dioxide 1.50
Azuki powder 59.40
Starch 6.50
Azuki bean extract 0.10
(Total) 100.00

  As can be understood from this result, the extract (antiallergic agent) derived from azuki bean broth of the present invention can be added to a wide variety of food types, and thus can be taken from a normal meal. Therefore, an effect for improving the quality of daily life (QOL) can be expected. The listed foods and beverages are examples. Naturally, the addition object of the extract (antiallergic agent) derived from the azuki bean broth of the present invention can be extended to other foods, beverages and other drugs. Further, the blending ratio is not limited to the disclosed amount, and can be adjusted as necessary.

  It was clarified that the extract of ethanol aqueous solution derived from azuki bean broth has antiallergic activity. Then, it came to obtain the azuki bean origin antiallergic agent. In particular, it is useful as an antiallergic agent that is effective in reducing type I allergic reactions. Furthermore, by creating a food to which the antiallergic agent is added, it can be ingested without difficulty from daily life.

Claims (4)

  After separating the supernatant from the broth produced by boiling azuki bean in hot water, adsorbing the supernatant to the synthetic adsorbent, adding an aqueous ethanol solution to the adsorbed synthetic adsorbent, and eluting the extract Azuki bean-derived antiallergic agent.   The azuki bean-derived antiallergic agent according to claim 1, wherein the ethanol aqueous solution is a 10% to 40% ethanol aqueous solution.   The azuki bean-derived antiallergic agent according to claim 1 or 2, wherein the extract exhibits activity against a type I allergic reaction.   Azuki bean-derived antiallergic agent-containing food, wherein the azuki bean-derived antiallergic agent according to any one of claims 1 to 3 is added to the food.

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